Abstract Description: In rheumatoid arthritis (RA), an inflammatory stimulus from secreted cytokines increases osteoclast activity, erodes bone surfaces, and progressively destroys joints. Research advances have identified the mechanism of osteoclastogenesis and bone resorption, and provided new medications to arrest disease progression. Despite this, there is little understanding of why eroded articular bone fails to heal. It is also unclear why patients with RA have systemic bone loss, currently attributed to increased osteoclastic bone resorption without evidence. We previously showed that tumor necrosis factor-1 (TNF), a major inflammatory mediator in arthritis, potently inhibits OB differentiation, suggesting that reduced OB activity abandons bone to unopposed osteoclastic resorption. In this proposal, we focus on the mechanism of reduced bone formation and healing in RA and hypothesize that TNF activates a key inhibitor of OB differentiation, the paired related homeodomain protein-1(Prx1). Prx1 potently suppresses OB differentiation and bone formation through inhibition of Osx and RUNX2 transcription. These factors are critical for the phenotypic commitment of pluripotent precursors to mature bone forming OB. We previously identified a TNF element in the Osx promoter that confers transcriptional inhibition. Activation of this inhibitory mechanism by TNF is signaled via MAPK. In preliminary studies, we utilized this TNF responsive DNA sequence as bait to identify Prx1 as the TNF-induced inhibitor of Osx transcription and confirmed Prx1 through mass spectroscopy and ChIP assay. RUNX2 transcription and mRNA can also be inhibited by Prx1. Prx1 is an embryonic limb bud enhancer active between E9.5 and E15. Our preliminary data will show that TNF reactivates Prx1 in adult bone and silencing of Prx1 completely abrogates TNF suppression of Osx and OB differentiation. Hypothesis: We hypothesize that Prx1 performs a pivotal function as a molecular signal of inflammatory suppression of bone healing and renewal. Aims / Methods: In this grant application we will evaluate the mechanism of Prx1 action and test our hypothesis in vivo using a skeletal-specific Prx1 knockout. Our hypothesis predicts that Prx1 deletion will confer resistance to TNF-induced bone loss in a murine model of rheumatoid arthritis. To test our hypothesis we propose three aims: Aim 1: To determine the mechanism of Prx1 action. 1A. Determine if Prx1 inhibition of Osx transcription requires MAPK and if Prx1 is phosphorylated. Specific inhibitors of MAPK will be used in cell culture. 1B. Determine the mechanism of action of Prx1on the Osx promoter: Determine if Prx1 interacts with RUNX2 at their continuous binding sites on the Osx promoter. Pull down experiments will be used to study protein-protein interaction. 1C. To determine if RUNX2 and/or Osx expression will abrogate Prx1 inhibition of OB differentiation. Expression vectors will be used in primary cell culture via lentivirus constructs to evaluate the effect of Osx or RUNX2 expression on Prx1 inhibition of OB differentiation. Aim 2: Determine if Prx1 is a mediator of TNF induced bone loss in vivo. 2A. Determine if OB-specific deletion of Prx1 increases baseline OB numbers and bone formation in adult animals. (Prx1 flox/flox X 2.3kb Cola1-Cre mice). Dynamic histomorphometry, microCT, histology, and bone density will be used. 2B. Determine if OB precursors cultured from Prx1 knockout mice are resistant to TNF inhibition of OB differentiation. Primary cell culture from marrow stromal cells will be used to test TNF responsiveness. Aim 3: Determine if Prx1 deletion ameliorates bone loss in TgTNF arthritic mice (Prxflox/flox, 2.3Cola1Cre X TgTNF). The effect of Prx knockout in bone on the extent of bone loss, erosions, and inflammation will be determined in the TNF expressing mouse. Techniques as in Aim 2A plus inflammation scores. Summary: This project will identify a novel pathway that prevents new bone formation, healing, and restoration of normal bone in inflammatory bone disorders, including RA, aging, and menopause.